Immunotherapy of cancer with allogeneic lymphocytes

ABSTRACT

Methods have been discovered for treating minimal residual disease following removal of most or a substantial fraction of malignant cells from a cancer patient. An autologous stem cell transplant is performed on the patient. Following partial hematopoiesis recovery, the patient is infused with allogeneic peripheral blood lymphocytes, either alone or in combination with in vivo or in vitro cytokine. The infused allogeneic lymphocytes engender an anti-malignant cell response and can be instrumental in prevention of disease relapse.

FIELD OF THE INVENTION

[0001] This invention relates to methods of eradicating residual tumorcells following surgical intervention, chemotherapy and/or radiotherapy.The methods involve autologous bone marrow transplantation followed byadministration of allogeneic peripheral blood leukocytes. Moreparticularly, this invention relates to use of HLA-compatible allogeneicperipheral blood lymphocytes (PBL), following autologous bone marrowand/or peripheral blood stem cell transplantation, to induce agraft-versus-malignant cell response. Such a response is instrumental inprevention of disease relapse.

BACKGROUND OF THE INVENTION

[0002] Patients with malignant hematological disorders resistant toconventional doses of chemotherapy and/or radiation may be treated byautologous or allogeneic bone marrow transplantation. Bone marrowtransplantation (BMT) makes it possible to administer to patients withresistant disease high, “supra-lethal,” combinations of chemotherapy andradiation, ignoring the irreversible toxicity of such therapeuticcombinations on the normal bone marrow compartment. Nevertheless, such“debulking” of a patient's tumor(s) can leave a fraction of residualmalignant cells that may lead to disease relapse. Several lines ofevidence suggest that a significant proportion of the beneficial effectof allogeneic BMT (i.e., BMT from an individual not geneticallyidentical to the host patient) stems from cell-mediated interactions ofimmune cells of donor origin against residual tumor cells in the hostthat have escaped the chemoradiotherapy debulking regimen.

[0003] Following allogeneic BMT, the incidence of relapse issignificantly lower in leukemia patients with clinical manifestations ofacute or chronic graft versus host disease (GVHD), as compared withpatients with no GVHD, indicating that immune-mediated allogeneicinteractions of immunocompetent cells of donor origin against the hostare also accompanied by graft vs. leukemia (GVL) effects. Weiden et al.,N. Engl. J. Med. 300: 1068 (1979); Weiden et al., N. Engl. J. Med. 304:1529-33 (1981); Weiden et al., Transplantation 13: 248-51 (1981);Barrett et al., Blood 74: 862 (1989); Sullivan et al., Blood 73: 1720(1989); Horowitz et al., Blood 75: 555 (1990); Slavin et al., BoneMarrow Transplant. 6: 155-61 (1990).

[0004] Higher relapse rates seem to occur in patients undergoingallogeneic BMT with T-lymphocyte depletion for prevention of GVHD,compared to recipients of non-T cell depleted marrow allografts,regardless of the severity of GVHD. Horowitz et al., Blood 75: 555(1990); Slavin et al., Bone Marrow Transplant. 6: 155-61 (1990); Goldmanet al., Ann. Inter. Med. 108: 806-14 (1988); Ringden and Horowitz,Transplant. Proc. 21: 2989-92 (1989); Goldman et al., Ann. Int. Med.108: 806 (1988). Likewise, relapse rates in patients with acute leukemiaor chronic myeloid leukemia reconstituted by bone marrow grafts obtainedfrom an identical twin (syngeneic grafts) are significantly higher thanin those reconstituted by bone marrow cells obtained from anHLA-identical but non-syngeneic sibling. Ringden and Horowitz,Transplant. Proc. 21: 2989-92 (1989). Similarly, relapse rates followingtransplantation of the patient's own (autologous) marrow, even followingadequate purging in vitro for elimination of residual leukemia cells,are significantly higher than following allogeneic BMT. Armitage, Curr.Opinion in Hematol. 1993: 240-45 (1993). Thus, the less-than optimalresults with autologous BMT (ABMT) are similar to the results seen withsyngeneic marrow transplantation. All of the above data suggests that inpractical terms GVHD or GVHD-potential correlates with a lower incidenceof relapse.

[0005] Allogeneic donor cells may also play a role against lymphoma, asshown in experimental animals, Slavin et al., Cancer Immunol.Immunother. 11: 155-58 (1981), and humans. Phillips et al., J. Clin.Oncol. 4: 480-88 (1986); Ernst et al., Proc. of the 4th InternationalConference on Lymphoma, Lugano 1990, Abstract #P35; Chopra et al., J.Clin. Oncol. 10: 1690-95 (1992). As shown in experimental animals,graft-versus-tumor effects (GVT), similar to graft-versus-leukemiaeffects (GVL), may occur following BMT, independently of GVHD.Moscovitch and Slavin, J. Immunol. 132: 997-1000 (1984).

[0006] Although GVHD-associated anti-leukemia effector mechanisms may beof benefit in BMT, nevertheless GVHD represents one of the majorobstacles in allogeneic- BMT, even among perfectly HLA-matched siblings.Acute GVHD develops in the majority of recipients of allogeneic BMT,with clinically significant manifestations in 26-64% of the recipientsdespite optimal post-transplant immunosuppressive prophylaxis. Storb etal., Blood 73: 1729 (1989). Chronic GVHD may occur in up to 45% of longterm survivors. Storb et al., Blood 73: 1729 (1989). There is nosatisfactory therapy for patients with established GVHD and hencepatients with severe manifestations of acute or chronic forms of thedisease are prone to develop multisystem complications that may requirefrequent hospitalizations, leading to poor quality of life andoccasionally serious or even fatal complications.

[0007] GVHD following allogeneic BMT can be prevented by adequatepretransplant T-lymphocyte depletion, using no post-transplant anti-GVHDprophylaxis. Reisner et al., In: Slavin, S (ed.), Tolerance in BoneMarrow and Organ Transplantation. Elsevier, Amsterdam (1984), p. ²⁹³;Waldmann et al., Lancet 2: 483-85 (1984); Slavin et al., Transplant.Proc. 17: 465-67 (1985). BMT without GVHD represents a better toleratedprocedure that may necessitate shorter hospitalization with superiorsubjective immediate outcome following allogeneic BMT. In addition, thequality of life of long-term survivors without GVHD is clearly betterthan for those patients with severe acute or chronic GVHD.

[0008] Unfortunately, the advantages of GVHD-free-allogeneic BMT arecounterbalanced by other serious complications due to untoward effectsof T-lymphocyte depletion, such as increased incidence of graftrejection, occurring in 10-30% of recipients, as well as increased ratesof tumor relapse. Martin et al., Bone Marrow Transplant. 3: 445 (1988);Kernan et al., Transplantation 43: 842 (1987); Waldmann et al., Lancet2: 483-85 (1984); Slavin et al., Transplant. Proc. 17: 465-67 (1985).Consequently, there seems to be no clear evidence to date for asignificant overall benefit of GVHD prevention by T-lymphocytedepletion.

[0009] Clearly, it would be a significant advance in the art to be ableto combine the benefits of minimal or controllable GVHD risk followingABMT or ASCT with induction of graft-versus-malignant cell response thatmay be associated with GVHD following allogeneic BMT.

SUMMARY OF THE INVENTION

[0010] The present invention includes a method of treating a humancancer patient who has undergone a malignant cell debulking regimen.Generally the patient is considered to be at risk for disease relapsedue to a population of residual malignant cells that may remain viablein the patient following the debulking procedure. A sample of stemcells, taken from the patient, is obtained and determined to be suitablefor autologous transplantation into the patient. This sample is used toperform an autologous transplant of the patient, i.e, infusing thepatient's stem cells back into the patient. The patient is thenmonitored until the patient is partially hematopoiesis recovered but isnot fully immune-reconstituted. Then, the patient is administered anHLA-compatible, allogeneic peripheral blood leukocyte preparation havinglymphocytes, in a regimen that causes a clinically significantgraft-versus-malignant cell response. The patient is then monitored forlevels of malignant cells deriving from any residual malignant cellsthat might have been present following the original debulking procedure.This monitoring may constitute one or more molecular or cellular assaysto detect or quantify malignant cells, may constitute a monitoringprogram to detect clinical signs of relapse, or any combination of thesemonitoring methods.

[0011] As used herein, a clinically significant response permits, forexample, the patient to avoid relapse, substantially prolongs the timeto relapse or otherwise engenders a beneficial condition thatsignificantly prolongs life. A graft-versus malignant cell response isnot clinically significant unless there is some clinical benefit to thepatient. Evidence for a clinically significant response may include, forexample, absence or delay of relapse, evidence for elimination ofminimal residual disease, i.e., elimination of disease-specific markersor, where appropriate, elimination of markers directed to host-specificcells.

[0012] The regimen for administration of an HLA-compatible leukocytepreparation including lymphocytes may comprise the following steps insequence:

[0013] a) treating the patient by administration (e.g., infusion) ofabout 10⁷ cells/kg to about 10⁹ cells/kg of HLA-compatible, allogeneicperipheral blood lymphocytes;

[0014] b) monitoring the patient for indications of agraft-versus-malignant cell response; and

[0015] c) if no or insufficient graft-versus-malignant cell responsedevelops in the patient, escalating the treatment by performing at leastone of the following procedures: (1) administration of a number ofHLA-compatible, allogeneic peripheral blood lymphocytes greater than thenumber of lymphocytes administered in step (a); (2) administration of anumber of HLA-compatible, allogeneic peripheral blood lymphocytes atleast as great as the number of lymphocytes administered in step (a),accompanied by administration of at least one T-cell-activating cytokineto said patient; (3) administration of HLA-compatible, allogeneiccytokine-activated lymphocytes (CAL) to said patient; and (4)administration of HLA-compatible, allogeneic CAL, accompanied byadministration in vivo of at least one T-cell-activating cytokine tosaid patient. More than one of these procedures can be performed if noor insufficient graft-versus-malignant cell response develops in thepatient following the first or subsequent procedure.

[0016] In an alternative embodiment, step (a) above can be augmented byadministering, concomitant with the allogeneic lymphocytes, at least oneT-cell-activating cytokine to the patient. Since the T-cell-activatingcytokine is administered directly to the patient, the infused allogeneiclymphocytes are exposed to the cytokine in vivo.

[0017] The present invention also includes an alternative method oftreating a human cancer patient who has undergone a malignant celldebulking regimen. Generally the patient is considered to be at risk fordisease relapse due to a population of residual malignant cells that mayremain viable in the patient following the debulking procedure. A sampleof stem cells, taken from the patient, is obtained and determined to besuitable for autologous transplantation into the patient. This sample isused to perform an autologous transplant of the patient, i.e, infusingthe patient's stem cells back into the patient. The patient is thenmonitored until the patient is partially hematopoiesis recovered but isnot fully immune-reconstituted. Then, the patient is administered anHLA-compatible, allogeneic peripheral blood leukocyte preparation havinglymphocytes, in a regimen that causes a mild graft-versus-host response.The patient is then monitored, as above, for levels of malignant cellsderiving from any residual malignant cells that might have been presentfollowing the original debulking procedure.

[0018] As used herein, the term “graft-versus-host response” includesbut is not limited to the classic clinical symptoms of graft-versus hostdisease (GVHD), known to those having ordinary skill in the art. Theterm “graft-versus-host response” also includes molecular or cellularresponses that correlate with the clinical symptoms of GVHD or with theimpending onset of the clinical symptoms of GVHD.

[0019] For the above-described alternative method, the regimen foradministration of an HLA-compatible leukocyte preparation includinglymphocytes may comprise the following steps in sequence:

[0020] a) treating the patient by administration of about 10⁷ cells/kgto about 10⁹ cells/kg of HLA-compatible, allogeneic peripheral bloodlymphocytes;

[0021] b) monitoring the patient for indications of a mildgraft-versus-host response; and

[0022] c) if no or insufficient graft-versus-host response develops inthe patient, escalating the treatment by performing at least one of thefollowing procedures: (1) administration of a number of HLA-compatible,allogeneic peripheral blood lymphocytes greater than the number oflymphocytes administered in step (a); (2) administration of a number ofHLA-compatible, allogeneic peripheral blood lymphocytes at least asgreat as the number of lymphocytes administered in step (a), accompaniedby administration of at least one T-cell-activating cytokine to saidpatient; (3) administration of HLA-compatible, allogeneic CAL to saidpatient; and (4) administration of HLA-compatible, allogeneic CAL,accompanied by administration in vivo of at least one T-cell-activatingcytokine to said patient. More than one of these procedures can beperformed if no or insufficient graft-versus-host response develops inthe patient following the first or subsequent procedure.

[0023] In an alternative embodiment, step (a) above can be augmented byadministering, concomitant with the allogeneic lymphocytes, at least oneT-cell-activating cytokine to the patient. Since the T-cell-activatingcytokine is administered directly to the patient, the infused allogeneiclymphocytes are exposed to the cytokine in vivo.

[0024] In an alternative embodiment, the regimen for administration ofan HLA-compatible leukocyte preparation including lymphocytes maycomprise the following steps in sequence:

[0025] a) administering to the patient about 10⁷ cells/kg to about 10⁹cells/kg of HLA-compatible, allogeneic peripheral blood lymphocytes andat least one T-cell-activating cytokine to the patient;

[0026] b) monitoring the patient for signs of a mild graft-versus-hostresponse;

[0027] c) if no or insufficient graft-versus-host response develops inthe patient, administering about 10⁷ cells/kg to about 10⁹ cells/kg ofHLA-compatible, allogeneic CAL and at least one T-cell-activatingcytokine to the patient;

[0028] d) monitoring the patient for signs of a mild graft-versus-hostresponse;

[0029] Alternatively, CAL may be given in the initial infusion. In thiscase the regimen for administration of an HLA-compatible leukocytepreparation including lymphocytes may comprise the following steps insequence:

[0030] a) administering to the patient about 10⁵ cells/kg to about 10⁹cells/kg of HLA-compatible, allogeneic peripheral blood lymphocytes inwhich at least some of the HLA-compatible, allogeneic peripheral bloodlymphocytes are CAL, and at least one T-cell-activating cytokine to thepatient;;

[0031] b) monitoring the patient for signs of a mild graft-versus-hostresponse;

[0032] c) if no or insufficient graft-versus-host response develops inthe patient, administering about 10⁵ cells/kg to about 10⁹ cells/kg ofHLA-compatible, allogeneic CAL and at least one T-cell-activatingcytokine to the patient;

[0033] d) monitoring the patient for signs of a mild graft-versus-hostresponse;

[0034] In any of the above described methods, the cytokine can be,without limitation, any one or more of the following: interleukin 2(IL2), interleukin 4 (IL4), interleukin 5 (IL5), interleukin 6 (IL6),interleukin 7 (IL-7), interferon alpha (IFNα), interferon gamma (IFNγ)and tumor necrosis factor (TNFα). Any of these cytokines can be a nativefactor obtained from natural sources, a factor produced by recombinantDNA methodology, a chemically synthesized polypeptide or other molecule,or any derivative having the functional activity of the native factor.

[0035] The stem cells used in the above-described methods may beobtained from bone marrow, from the peripheral circulation, or, whereappropriate, from fetal sources such as fetal tissue, fetal circulationand umbilical cord blood. Cancer patients treatable with the methods ofthe present invention are any patients having a pathological conditioncaused by malignant cells, including without limitation leukemia,lymphoma, and breast cancer. The HLA-compatible allogeneic cellsemployed in the present invention preferably are fully HLA-matched withthe patient. Alternatively the allogeneic cells may be at leasthaploidentical with the patient. If the allogeneic cells are derivedfrom a sibling of the patient, the cells preferably are fully HLAmatched with the patient, although some mismatch may be tolerated. Forexample, the allogeneic cells from a sibling to the patient may, in somecases, be single HLA locus-mismatched. If the allogeneic cells arederived from an unrelated individual, preferably the cells are fully HLAmatched with the patient.

[0036] The present invention further includes an article of manufacturecomprising packaging material and a biological cell container within thepackaging material. The packaging material contains a label or packageinsert indicating that the biological cell container, and/or anycontents therein, are to be used in any of the above-described methodsfor treating a human cancer patient.

BRIEF DESCRIPTION OF THE FIGURES

[0037]FIG. 1 depicts the results of adoptive transfer of spleen cellsobtained from lethally irradiated F1 mice transplanted with 10⁷syngeneic bone marrow cells and 10⁵ BCL1 cells in addition to 20-30×10⁶PBL from allogeneic mice with (A, n=40) or without (B, n=40) concomitantin vivo rhIL2 treatment (12×10⁴ IU×2/day for 5 days IP). A similar groupreceived syngeneic (F1) PBL given with (C, n=20) or without (D, n=20)rhIL2. A control group had adoptive transfer of spleen cells obtainedfrom 10 untreated F1 recipients (E, n=20).

[0038]FIG. 2 depicts the results of adoptive transfer of spleen cellsobtained from lethally irradiated F1 mice reconstituted with 10⁷syngeneic bone marrow cells mixed with 10⁵ BCL1 cells. Cell-mediatedimmunotherapy consisted of intravenous administration of increasingnumbers of C57BL/6 spleen cells: 1×10⁶ (A, n=10); 3×10⁶ (B, n=10);10×10⁶ (C, n=10) and 30×10⁶ (D, n=10). One of three experiments isshown.

[0039]FIG. 3 depicts results of adoptive transfer of spleen cellsobtained from BALB/c mice treated with cyclophosphamide (300 mg/kg IP),inoculated 24 hours later with 10³ BCL1 cells, and one day laterreceiving 4×10⁶ syngeneic, ASTA-Z-treated bone marrow cells.Immunotherapy consisted of a mixture of 20×10⁶ allogeneic C57BL/6 spleenand lymph node cells either with (A, n=6) or without (B, n=6) rhIL2treatment in vivo (12×10⁴ IU×3/day for 3 days IP). Recipients of amixture of 20×10⁶ syngeneic BALB/c spleen and lymph node cells treatedwith rhIL2 (C, n=7) and recipients of 10³ BCL1 cells only (D, n=10)served as controls.

[0040]FIG. 4 depicts the results of adoptive transfer of spleen cellsobtained from lethally irradiated F1 mice inoculated with 10⁵ BCL1 cellsand 30×10⁶ bone marrow cells pre-activated in vitro for 4 days withrhIL2; mice with no additional treatment (A, n=33), mice with in vivorhIL2 treatment (12×10⁴ IU×2/day for 5 days IP) (B, n=25); controls:recipients of 10⁵ spleen cells obtained from untreated control miceinoculated with 10⁵ BCL1 cells (C, n=30).

DETAILED DESCRIPTION OF THE INVENTION

[0041] The present inventor has employed allogeneic peripheral bloodlymphocytes, alone or in combination with cytokine treatment in vivo orin vitro, for successful elimination of minimal residual diseasefollowing chemotherapy and/or radiotherapy. Appropriate treatmentregimens have been circumscribed by studies in laboratory animals, andthe treatment protocols have been further extended to human patients athigh risk for disease relapse.

[0042] A spontaneous, transplantable murine B-cell leukemia/lymphoma ofBALB origin (BCL1) was used to investigate elimination of minimalresidual disease (MRD) following bone marrow transplantation. As usedherein, MRD refers to a condition wherein residual malignant cellsremain viable in a patient following a primary and/or metastatic tumor“debulking” regimen. The debulking regimen may include surgicalexcision, chemotherapy or radiotherapy, or any combination of theseapproaches. Such treatment removes a significant fraction of malignantcells from the patient, but may leave a clinically significant number ofresidual malignant cells that put the patient at risk of relapse. Theterm “tumor” as used herein includes all pathological conditionsinvolving malignant cells; this can include “solid” tumors arising insolid tissues or organs as well as “liquid” tumors such as leukemias andlymphomas deriving from malignant transformation of hematopoietic cells.

[0043] In autologous bone marrow transplantation (ABMT) with humanpatients, an individual receives his or her own bone marrow cells byinfusion following a tumor debulking procedure. Generally, the bonemarrow cells are taken from the patient and preserved, for example bycryopreservation, prior to the debulking procedure. This permits anotherwise lethal debulking regimen to be employed, e.g., chemotherapy orradiotherapy that severely damages or destroys the patient's bonemarrow. Following the debulking procedure, the patient's bone marrow isreconstituted by stem cells present in the preserved sample of bonemarrow.

[0044] Stem cells capable of reconstituting a patient's immune systemcan be obtained not only by direct extraction from the bone marrow, butalso from the patient's peripheral circulation following mobilization ofsuch cells from the bone marrow. This can be accomplished by treatmentof the patient with granulocyte colony stimulating factor (G-CSF) orother appropriate factors that induce movement of stem cells from thebone marrow into the peripheral circulation. Following-mobilization, thestem cells can be collected from peripheral blood by any appropriatecell apheresis technique, for example through use of a commerciallyavailable blood cell collection device as exemplified by the CS 3000®Plus blood cell collection device marketed by the Fenwal Division ofBaxter Healthcare Corporation. Methods for performing apheresis with theCS 3000® Plus machine are described in Willams et al., Bone MarrowTransplantation 5: 129-33 (1990) and Hillyer et al., Transfusion 33:316-21 (1993), both publications being incorporated herein by reference.Stem cells collected from the peripheral blood are termed herein“peripheral blood stem cells” (PBSC). The term “autologous stem celltransplantation” (ASCT) is used herein to refer to any infusion into apatient of that same patient's stem cells, derived from any appropriatesource (e.g., bone marrow or peripheral circulation). As such, ABMT,where the autologous infused cells are extracted directly from the bonemarrow of the patient, may be considered simply one form of ASCT.

[0045] It is possible to create an experimental regime in mice thatsimulates ASCT in humans. This is done through use of stem cell donorsand recipients derived from a syngeneic strain of mice. In such strains,inbreeding has created a situation in which, for practical purposes,mice within the strain are genetically identical to each other. Suchmice accept tissues and organs transplanted between individuals withoutevidence of immune rejection, in a manner analogous to acceptance of apatient's own cells following ASCT. Transplantation of bonemarrow-derived stem cells between such mice is referred to herein as“syngeneic bone marrow transplantation” (SBMT) and should be consideredanalogous to ABMT and ASCT in humans.

[0046] In the present experiments, mice received a lethal dose of totalbody irradiation (TBI) or, alternatively, a lethal dose ofcyclophosphamide administered intraperitoneally. Bone marrow cells (BMC)were extracted directly from the bone marrow of syngeneic mice. In somecases, these BMC preparations were treated with mafosfamide (ASTA-Z) tosimulate a “purging” procedure in which the patient's stem cells, priorto ASCT, are treated to remove or destroy at least a fraction of anycontaminating malignant cells. One day after irradiation or treatmentwith cyclophosphamide, the mice received 10⁷ syngeneic bone marrow cellsby infusion into the lateral tail vein. To simulate MRD, 10⁵ BCL1 tumorcells were added to the syngeneic BMC prior to SBMT.

[0047] Following SBMT, recipient mice received either allogeneiccell-mediated immunotherapy (Allo-CMI) or allogeneic cell-mediatedcytokine-activated immunotherapy (Allo-CCI). Allo-CMI involved transferof immunocompetent allogeneic lymphocytes (i.e., lymphocytes from amouse strain other than that of the recipient mice) at various times andat various doses, administered post-SBMT. Generally these lymphocytesrepresented peripheral blood lymphocytes (PBL) or mixtures of donorspleen and lymph node cells. Allo-CCI involved transfer of allogeneiclymphocytes pre-activated in vitro with recombinant human interleukin 2(rhIL2). As used herein, the term “CAL” refers to such“cytokine-activated lymphocytes.” In some experimental protocols, theAllo-CMI or Allo-CCI regimen was accompanied by simultaneous in vivoadministration of rhIL2 to the recipient mice, in order to facilitateadditional activation of the infused lymphocytes in vivo.

[0048] Failure to develop leukemia in primary recipients does not proveelimination of all BCL1 cells, since active suppression of existingtumor cells can prevent development of overt disease in these animals.This has been documented following allogeneic BMT and rhIL2 therapy.Slavin et al., Cancer Immunol. Immunother. 11: 155 (1981); Slavin etal., Nat. Immun. Cell Growth Regul. 7: 180 (1988). To establishconclusive evidence for eradication of residual malignant cells, spleencells from the treated, or recipient, mice were adoptively transferredto secondary syngeneic recipients. If these secondary recipients failedto develop leukemia, it was judged that the original Allo-CMI- orAllo-CCI-treated mice were free of viable malignant cells, since as fewas 1-10 cells have been shown to be capable of causing disease. Slavinet al., Cancer Res. 41: 4162 (1981); Cohen et al., J. Immunol. 151:4803-10 (1993).

[0049] The results of the SBMT experiments with mice suggested thateffective immunotherapy of MRD can be achieved in vivo by cell therapywith alloreactive lymphocytes through an effect that can be furtherenhanced in vivo with a short course of intermediate-dose rhIL2.GVL-like effects were also induced by infusion of CAL without causingany gross impairment of the hematopoietic capacity of BMC in lethallyirradiated recipients. Moreover, GVL effects induced by allogeneiclymphocytes as well as CAL could be further enhanced by concomitantcondition of stabilized blood counts) is attained, as indicated byacceptable levels of, for example, white blood cells (WBC), hemoglobin(Hb) and platelets. This condition of partial hematopoiesis recovery maybe achieved in a matter of weeks following ASCT in humans, well beforefull immune reconstitution diminishes the likelihood of successfulAllo-CMI and/or Allo-CCI.

[0050] The Allo-CMI and Allo-CCI strategies developed in mice have beenadapted to human patients in a variety of protocols undertaken by thepresent inventor in treating cancer patients at high risk of diseaserelapse. Cancer patients having acute myelogenous leukemia, chronicmyelogenous leukemia, non-Hodgkin's lymphoma and metastatic breastcancer have been treated with the methods of the present invention.

[0051] Two of the earlier-treated patients with acute myelogenousleukemia were given relatively low numbers (e.g., about 10⁴ cells/kg) ofallogeneic peripheral blood lymphocytes as a first dose, in an effort tominimize the risk of serious GVHD. Thereafter, the patients were givenescalating numbers of allogeneic peripheral blood lymphocytes at varioustime intervals to increase the chances of clinically significantgraft-versus malignant cell response. Both of these early patients(patients 1 and 2 in Example 2, set out below) relapsed and died.Following this experience, it was apparent that administration of gradedincrements of PBL, beginning with relatively low numbers, may not beeffective. It was hypothesized that the initial low dose of allogeneicPBL produced only immunization without significant GVHD orgraft-versus-malignant cell response. Thus, later, higher doses rhIL2therapy in vivo, most likely due to continuous in vivo activation ofallogeneic effector cells against residual host malignant cells.

[0052] The data further indicate that eradication of BCL1 can beaccomplished before overt clinical manifestations of GVHD in the primaryrecipients would have occurred, since experiments showed that GVL-likeeffects against BCL1 cells were achieved within 1-2 weeks followingadministration of allogeneic lymphocytes. This implies that temporaryengraftment of allogeneic effector cells may be sufficient to inducebeneficial GVL effects against MRD, without the need for permanentresidence of allogeneic effector cells, which may put the patient atrisk for severe GVHD across major histocompatibility barriers. Moreover,as the time interval between ASCT and the Allo-CMI/CCI treatmentincreases, larger numbers of donor's PBL can be administered with lesslikelihood of severe GVHD. Slavin et al., J. Exp. Med. 147: 963 (1978);Slavin et al., Blood 80: 535a (1992).

[0053] As such, it is useful to administer an Allo-CMI or Allo-CCIregimen after the patient is partially hematopoiesis recovered followingthe original tumor debulking procedure/ASCT. This raises the likelihoodthat the allogeneic inoculum will be rejected by reconstituting hostimmune cells in due time. On the other hand, it is also desirable toundertake the Allo-CMI/Allo-CCI regimen prior to full immunereconstitution of the patient, since the likelihood of prematurerejection of the allogeneic inoculum, prior to beneficial GVL/GVTeffects, is thereby reduced. Since full immune reconstitution followingASCT in humans frequently requires up to one year, the patient can bemonitored until a stable clinical condition (e.g., of allogeneic PBL,which otherwise might be effective, are promptly rejected by the patientand rendered ineffectual for graft-versus-malignant cell activity.Therefore, in subsequent cases, more cells (e.g., at least about 10⁷cells/kg) were administered in the initial dose. With this initialhigher dose of allogeneic cells following ASCT, satisfactory resultshave been obtained in many patients within the treatment population todate.

[0054] Allogeneic cells preferably are chosen from human leukocyteantigen (HLA)-compatible donors. Generally, HLA-compatible lymphocytesmay be taken from a fully HLA-matched relative such as a parent, brotheror sister. However, as shown with patient No. 12 in Example 2, below,donor lymphocytes may be sufficiently HLA-compatible with the recipientto obtain the desired result even if a sibling donor is single-locusmismatched. If a donor is unrelated to the recipient, preferably thedonor lymphocytes are fully HLA matched with the recipient.

[0055] In a preferred embodiment of the present invention, an initialdose of at least about 10⁷ HLA-compatible allogeneic lymphocytes/kg isgiven once the patient has achieved a clinically stable condition (e.g.,stabilized blood counts). Preferably, the patient is also administeredcytokine in vivo concomitant with administration of the HLA-compatiblelymphocytes,. Preferably the cytokine is rhIL2 at a dose of about 6×10⁶IU of rhIL2/m²/day, by subcutaneous injection, beginning on the same dayas infusion of the allogeneic lymphocytes. Other appropriate cytokinesmay be used alone or in combination with rhIL2, as long as the desiredenhancing effect is obtained. Such additional cytokines include, withoutlimitation, native or recombinant forms of IL6, IL7 and alpha andgamma-interferon (INFα, INFγ, respectively).

[0056] If GVHD fails to develop in a patient given these numbers ofHLA-compatible allogeneic lymphocytes with concomitant in vivo cytokine,then the treatment regimen is escalated. Preferably this is done byadministering a second dose of allogeneic HLA-compatible lymphocytespreactivated in vitro with cytokine. Preferably the cytokine is rhIL2,although other appropriate cytokines may be used alone or in combinationwith rhIL2, as long as the desired activation in vitro is obtained. Suchadditional cytokines include, without limitation, native or recombinantforms of IL6, IL7 and alpha-interferon (INFα), alone or in combinationwith IL2.

[0057] Prior to administration of the second dose of cells comprisingCAL, and contingent on the particular status of an individual patient,cyclophosphamide (Cytoxan) or other appropriate immunosuppressants maybe administered to the patient to avoid rejection of the second dose ofcells. That is, the immunosuppressant is given in a dose effective forkilling host T cells that might otherwise operate to reject the secondallogeneic inoculum; the immunosuppressant may have the added benefit ofeliminating potential host suppressor cell functions that can interferewith the GVT effects of the infused CAL. Preferably, in vivo cytokine isadministered to the patient concomitant with the second dose of cells(CAL).

[0058] Although the above-described preferred embodiment is presentlyrecommended, it is to be understood that any combination of allogeneicperipheral blood lymphocytes, in vivo cytokine and/or CAL is covered bythe present invention, so long as the initial dose of cells is capableof eliciting a beneficial GVT response. This dose of cells correspondsto a number of HLA-compatible allogeneic peripheral blood lymphocytesthat elicits a host response beyond mere immunization to a second doseof cells from the same or similar donor. Generally, this numbercorresponds to at least about 10⁷ allogeneic peripheral bloodlymphocytes/kg. However, it is possible that a lower dose of cells,e.g., about 10⁵ cells/kg, could be used if, for example, CAL were usedfor the initial infusion.

[0059] Between the Allo-CMI/CCI treatments or at the conclusion of anAllo-CMI/CCI regimen, the patient may be monitored for levels ofmalignant cells, i.e., for evidence of minimal residual disease. Suchmonitoring may comprise patient follow-up for clinical signs of relapse.The monitoring may also include, where appropriate, various molecular orcellular assays to detect or quantify any residual malignant cells. Forexample, in cases of sex-mismatched donors and recipients, residualhost-derived cells may be detected through use of appropriate sexmarkers such as Y chromosome-specific nucleic acid primers or probes. Incases of single HLA locus mismatches between donors and recipients,residual host cells may be documented by polymerase chain reaction (PCR)analysis of Class I or Class II loci that differ between the donor andrecipient. Alternatively, appropriate molecular markers specific fortumor cells can be employed. For example, nucleic acid primers and/orprobes specific for the bcr/abl translocation in chronic myelogenousleukemia, for other oncogenes active in various tumors, for inactivatedtumor suppressor genes, other tumor-specific genes, or any other assayreagents known to be specific for tumor cells, may be employed. Any ofthese or functionally comparable procedures may be used to monitor thepatient for evidence of residual malignant cells.

[0060] Under normal circumstances, recipients of autologous orallogeneic bone marrow grafts receive only irradiated blood productswhen such products are required by the patient. These products areirradiated in order to avoid the possibility of engraftment ofimmunocompetent T-lymphocytes derived from the donor's blood product(e.g., platelets or red blood cells). In most institutions, irradiatedblood products are also used for patients receiving high doseconventional chemotherapy without transplant, e.g., blood products givenfollowing induction of remission in leukemia and lymphoma patients.Obviously, the chances for engraftment of immunocompetent T-lymphocytesfrom otherwise mismatched blood products is relatively small undernormal circumstances. However, if immunosuppression is sufficient topermit engraftment, GVHD can be “stormy” and lethal.

[0061] In the methods of the present invention, non-irradiateddonor-type lymphocytes are used intentionally for induction ofgraft-versus-malignant cell effects. The methods are structured toproduce transient engraftment, so as to induce graft-versus-malignantcell effects that may be accompanied by mild GVHD. Since the donor cellsused in the Allo-CMI and Allo-CCI procedures are HLA-compatible with therecipient, chances of engraftment are better than if the donor's cellswere not functionally matched with the patient's majorhistocompatibility complex. Moreover, the chance of immediate rejectionon the one hand and lethal GVHD on the other hand are relatively smallbecause of the HLA compatibility. As such, the Allo-CMI/CCI protocols ofthe present invention provide the possibility for transient engraftmentof donor's PBL with effective GVT and with a minimal chance forinduction of severe GVHD.

[0062] The invention will be further understood with reference to thefollowing illustrative embodiments, which are purely exemplary, andshould not be taken as limiting the true scope of the present inventionas described in the claims.

EXAMPLE 1 Syngeneic BMT (SBMT) in Mice Followed by Allo-CMI or Allo-CCI

[0063] I. Methods

[0064] A. Mice.

[0065] BALB/c (BALB), and C57BL/6 (C57), (BALB/c x C57BL/6)F1 (F1) mice,2-6 months old, were purchased from the breeding colony of the HebrewUniversity-Hadassah Medical School, Jerusalem. Mice were kept understandard conditions, with acidic water (pH 2.7) and no specialprotective measures. Mice were given 0.5% neomycin sulfate in theirdrinking water for 2 weeks post-transplantation.

[0066] B. Murine B-Cell Leukemia (BCL1).

[0067] BCL1, a spontaneous, transplantable B-cell leukemia/lymphoma ofBALB origin is characterized by marked (up to 50 fold) splenomegaly,accompanied by extreme peripheral blood lymphocytosis (>200,00/mm³) andresults in death of all mice inoculated with >10-100 tumor cells. Slavinet al., Nature 272: 624 (1978); Slavin et al., Cancer Res. 41: 4162(1981). BCLI was maintained in vivo in BALB mice by IV passage of10⁶-10⁷ peripheral blood lymphocytes (PBL) obtained from tumor bearingmice. Mice with marked lymphocytosis in the blood were subsequently usedas BCL1 cell donors for experimental mice. PBL counts for allexperimental groups were carried out weekly. Leukemia was defined as PBLcounts exceeding 20,000/mm³. At the peak of disease PBL counts usuallyreached >100,000/mm³.

[0068] C. Mafosfamide (ASTA-Z).

[0069] ASTA-Z was kindly provided by Drs M. Peukert and H. Sindermann(Astawerke, Bielefeld, Germany) as a lyophilized powder and was freshlydissolved in saline before use. ASTA-Z has been employed in vitro toreduce or eliminate malignant cell populations from bone marrowpreparations. Douay et al., CR Acad. Sci. Paris t. 301, Ser III, no.6:303 (1985).

[0070] D. Conditioning with Radiation and Cyclophosphamide Prior to BMT.

[0071] Mice were exposed to a single dose of 750 cGy total bodyirradiation (TBI) from a Philips X-ray unit (250 kV 20 mA) with a focusto skin distance of 70 cm at a dose rate of 60 cGy/min. Alternatively,mice were conditioned with freshly dissolved cyclophosphamide (CY) (300mg/kg) (Taro, Israel) given intraperitoneally (IP). Twenty-four hourslater, mice received 10⁷ syngeneic marrow cells via the lateral tailvein.

[0072] E. Preparation of Bone Marrow Cells (BMC).

[0073] BMC were obtained from the femura, tibiae and humera of syngeneicmice. Mononuclear cells containing 10⁷ BMC in 0.25 ml Hank's medium wereinjected into the lateral tail vein of recipients 24 hourspost-radiation.

[0074] F. Purging Procedure.

[0075] Cells were resuspended at a concentration of 20×10⁶ cells/ml inHank's medium containing 4% human serum albumin. ASTA-Z was then addedto a final concentration of 100 μg/ml. Both untreated control cells andASTA-Z treated BMC were incubated at 37° C. for 30 minutes, washed twicein Hank's medium and counted. Purged or unpurged BMC (4×10⁶) wereinjected into BALB mice conditioned with CY.

[0076] G. Recombinant Human Interleukin-2 (rhIL2).

[0077] rhIL2 provided as 1 mg Proleukin (3×10⁶ Cetus Units, equivalentto 18×10⁶ International Units) was kindly supplied by Dr. S. L.Aukerman, Cetus/Chiron, Calif., USA. rhIL2 was initially diluted withwater for injection and subsequently rediluted with dextrose 5%.International units (IU) are used throughout the remainder of thepresent application.

[0078] H. Activation of BMC by rhlL2.

[0079] BMC were cultured in 225 cm³ flasks (Corning 25160-225, CorningGlass, Corning N.Y.) in RPMI 1640 medium (Beit Haemek, Israel)containing L-glutamine, non-essential amino acids, pyruvate, 10% bovinecalf serum (BCS) and rhIL2 (6,000 IU/ml) for 4 days in a humidifiedincubator with 5% CO₂ at 37° C. Following harvesting, viability wasdetermined by the trypan blue exclusion method.

[0080] I. Simulation of Minimal Residual Disease Following SyngeneicBone Marrow Transplantation.

[0081] In order to simulate minimal residual disease (MRD)quantitatively, 10⁵ BCL1 cells were added to the marrow inoculum duringsyngeneic bone marrow transplantation (SBMT), prior to immunotherapy.

[0082] J. Immunotherapy by Immunocompetent Allogeneic Lymphocytes.

[0083] Allogeneic cell-mediated immunotherapy (Allo-CMI) consisted ofadoptive transfer of immunocompetent allogeneic lymphocytes (PBL or amixture of donor spleen and lymph node cells) as detailed in the resultsfor each experiment, below. Allogeneic cell-mediated cytokine-activatedimmunotherapy (Allo-CCI) consisted of adoptive transfer of allogeneiclymphocytes pre-activated in vitro with rhIL2 (allogeneic “CAL”) In someexperiments allogeneic lymphocyte infusion was followed by subsequent invivo activation with rhIL2, by additional Allo-CMI with in vivo rhIL-2,or by additional Allo-CCI with in vivo rhIL-2, respectively.

[0084] K. Detection of Residual Clonogenic BCL1 by Adoptive TransferExperiments.

[0085] In order to determine whether or not residual BCLI cells werepresent after various treatments, 10⁵ spleen cells obtained from treatedmice were adoptively transferred to untreated secondary syngeneic (BALB)recipients. Absence of leukemia (>100 days) in secondary recipients wasindicative of elimination of BCL1 since as few as 1-10 cells werepreviously shown to cause disease.

[0086] L. Statistical Analysis.

[0087] The significance of differences between treated and untreatedmice was calculated by the independent statistical t-test.

[0088] II. Results

[0089] A. Induction of Allo-CMI and Allo-CCI Effects.

[0090] F1 mice were lethally irradiated (750 cGy) and transplanted with10⁷ syngeneic BMC. Following inoculation of 10⁵ BCL1 cells to simulateMRD, varying numbers of C57 PBL were administered intravenously toinduce GVL-like effects through allo-CMI. In order to detect theefficacy of allo-CMI in eradicating residual BCL1 cells, aliquots of 10⁵spleen cells pooled from 2-3 experimental mice were adoptivelytransferred to secondary normal BALB recipients, one or two weekspost-SBMT.

[0091]FIG. 1 summarizes results obtained from three differentexperiments in a total of 120 mice. Injection of 20-30×10⁶ PBL, obtainedfrom C57 mice to induce allo-CMI after SBMT in F1 recipients,effectively eliminated residual BCL1 cells, as none of 40 secondaryadoptive BALB recipients developed leukemia (>180 days). In contrast,leukemia developed in all 20 secondary BALB recipients inoculated with10⁵ spleen cells obtained from Fl recipients that had received 20-30×10⁶PBL from syngeneic donors post-SBMT. Addition of rhIL2 (12×10⁴ IU×2/dayfor 5 days IP) post-transplant did not improve the disease-free survivalof secondary recipients of 10⁵ spleen cells (obtained from similarlytreated F1 mice) since all 20 secondary recipient BALB mice developedleukemia (FIG. 1). Addition of rhIL2 in vivo at the same dose torecipients of 20×10⁶ allogeneic PBL for further in vivo activation ofeffector cells did not induce measurable additional GVL effects sinceall 40 secondary BALB recipients remained disease free (>180 days) (FIG.1).

[0092] B. Quantitative Effect of the Number of Effector Cells on theEfficacy of Allo-CMI.

[0093] Anti-leukemic effects mediated by allo-CMI were cell-dosedependent. As shown in FIG. 2, all SBMT recipients injected with 30×10⁶C57 spleen cells completely resisted the development of leukemiafollowing inoculation of 10⁵ BCL1 cells. Injection of 10×10⁶ allogeneicspleen cells together with 10⁵ BCL1 cells induced effective allo-CMI in70% of the secondary adoptive recipients. However, reduction of allo-CMIinducing C57 spleen cells to 3×10⁶ or 1×10⁶ failed to eliminate residualBCL1 cells and all secondary adoptive recipients developed leukemia(FIG. 2).

[0094] C. Induction of Allo-CMI and Allo-CMI/IL-2 Effects FollowingTransplantation with ASTA-Z-Purged BMC.

[0095] The feasibility of induction of allo-CMI was investigated byconditioning with high-dose CY followed by rescue of recipients withASTA-Z-purged syngencic BMC. BALB recipients received high-dose CY (300mg/kg IP) and were injected 24 hours later with 10³ BCL1 cells tosimulate MRD. One day later, all mice received intravenously 4×10⁶ASTA-Ztreated syngeneic BMC. Mice were divided into 3 experimental groups: thefirst group (6 mice) received intravenously a mixture of allogeneic C57spleen and lymph node cells (20×10⁶ cells) for induction of allo-CMI;the second group (6 mice) received identical cell therapy withadditional in vivo potentiation of GVL by rhIL2 treatment (12×10⁴IU×3/day for 3 days, IP); the third group (7 mice) received a mixture ofsyngeneic spleen and lymph node cells, with an identical in vivo rhIL2treatment. One week later, aliquots of 10⁵ cells from a pool of 2-3spleens obtained from each experimental group were adoptivelytransferred to secondary BALB mice.

[0096] As shown in FIG. 3, all mice inoculated with spleen cells fromcontrol mice given 10³ BCL1 cells, or mice given syngeneic BALBlymphocytes with in vivo rhIL2, developed leukemia and died within 40and 60 days, respectively. Likewise, secondary recipients of 10⁵ spleencells obtained from mice that were treated with allo-CMI, usingallogeneic C57 cells alone, showed no measurable GVL effects since allrecipients developed leukemia. In contrast, addition of rhIL2 in vivofollowing administration of C57 spleen and lymph node cell mixturesinduced substantial anti-leukemic effects and 50% of the secondaryadoptive recipient mice remained leukemia-free for >125 days (FIG. 3).

[0097] D. Enhancement of Immunotherapeutic Effect by in vitro Activationof Allogeneic lymphocytes with rhIL2.

[0098] The following experiment was designed to test for potentialenhancements in efficacy of treatment by in vitro pre-activation ofallogeneic effector cells with rhIL2. Lethally irradiated (750 cGy TBI)F1 mice were infused with 30×10⁶ C57 BMC pre-activated in vitro for 4days with rhIL2. BMC were mixed with 10⁵ BCL1 cells to simulate MRD.Results of 3 separate sets of experiments gave similar results andtherefore the data were pooled (FIG. 4).

[0099] All F1 recipients were divided into two groups. The first groupof 33 mice received no additional treatment. Mice in the second group(25 mice) were injected with rhIL2 (12×10⁴ IU×2/day for 5 days, IP) inan attempt to further increase efficacy of cell therapy by continuousactivation of rhIL2-dependent effector cells in vivo. Aliquots of 10⁵cells obtained from a pool of spleen cells prepared from mice of eitherexperimental group were adoptively transferred to secondary BALBrecipients. As shown in FIG. 4, 10 of 33 secondary recipients of spleencells obtained from the first experimental group remained disease-freefor >150 days. Additional in vivo rhIL2 therapy in the secondexperimental group further improved the Allo-CCI effects, as 19 of 25secondary recipients remained disease-free for an observationperiod >150 days (p=0.05) (FIG. 4).

EXAMPLE 2 Autologous Stem Cell Transplantation (ASCT) in Humans Followedby Allo-CMI and/or Allo CCI

[0100] I. Patient Treatment Protocols

[0101] Patient No. 1.

[0102] This female patient was diagnosed with acute myclogenous leukemia(AML), French American British (FAB) classification M4, and was in firstcomplete remission (i.e., no evidence of disease) at the time ofautologous stem cell transplantation (ASCT). The patient was 41 yearsold at the time of ASCT. Prior to ASCT, she received a conditioningregimen of Busulfan, 4 mg/kg, days 6 through 9 pre-ASCT (days −9 to −6),as well as Cytoxan (cyclophosphamide), 50 mg/kg, days −5 to −2,Cotrimoxazol, 10 mg/kg, days −10 to −2, Allopurinol, 300 mg/kg, days −10to −1 and cytosine arabinoside (Ara-C), 25 mg intrathecally.

[0103] Prior to ASCT, the autologous cells to be infused were purged bytreatment with Mafosfamide (ASTA-Z). ASTA-Z was provided by Drs. M.Peukert and H. Sindermann (Astawerke, Bielefeld, Germany) as alyophilized powder and was freshly dissolved in saline before use.Autologous cells were resuspended at a concentration of 20×10⁶ cells/mlin Hank's medium containing 4% human serum albumin. ASTA-Z was thenadded to a final concentration of 100 ug/ml and the cells were incubatedin the ASTA-Z at 37° C. for 30 min. After this, the cells were washedtwice in Hank's medium and counted. Cells were cryopreserved and kept inliquid nitrogen until used. ASCT consisted of 2.5×10⁸ nucleated bonemarrow cells/kg, infused intravenously (IV) on day 0.

[0104] On day 1 following ASCT (day +1), the patient received 10⁴ Tcells/kg of peripheral blood lymphocytes (PBL) from an HLA-matcheddonor. On days +3, +22, +29 and +36 she received PBL from the same donorat an equivalent dose of 10⁵, 10⁵, 10⁶, and 10⁶ T cells/kg,respectively. On day +47 she received PBL from the same donor at anequivalent dose of 10⁷ T cells/kg. The patient showed no evidence ofGVHD.

[0105] Patient No. 2.

[0106] This female patient was diagnosed with AML, FAB M5, and was infirst complete remission at the time of ASCT. The patient was 42 yearsold at the time of ASCT. Prior to ASCT, she received a conditioningregimen of Busulfan, 4 mg/kg, (days −9 to −6), as well as Cytoxan, 50mg/kg, days −5 to −2, Cotrimoxazol, 10 mg/kg, days −10 to −2,Allopurinol, 300 mg/kg, days −10 to −1 and Ara-C, 25 mg intrathecally.

[0107] Prior to ASCT, the autologous bone marrow cells to be infusedwere purged by treatment with ASTA-Z. Autologous cells were resuspendedat a concentration of 20×10⁶ cells/ml in Hank's medium containing 4%human serum albumin. ASTA-Z was then added to a final concentration of100 ug/ml and the cells were incubated in the ASTA-Z at 37° C. for 30min. After this, the cells were washed twice in Hank's medium, countedand cryopreserved. ASCT consisted of 1×10⁸ nucleated bone marrowcells/kg, infused IV on day 0.

[0108] On day +1, the patient received 10⁴ T cells/kg of PBL from anHLA-matched donor. On days +8, +18 and +26 she received PBL from thesame donor at an equivalent dose of 10⁵, 10⁶, and 10⁷ T cells/kg,respectively. On day +80 she received PBL from the same donor at anequivalent dose of 10⁷ T cells/kg, with 3×10⁶ IU of rhIL-2/m²/day for 3days, by subcutaneous injection beginning on day +80. The patient showedno evidence of GVHD.

[0109] Patient No. 3.

[0110] This female patient was diagnosed with AML, FAB M3, and was infirst complete remission at the time of ASCT. The patient was 32 yearsold at the time of ASCT. Prior to ASCT, she received a conditioningregimen of Busulfan, 4 mg/kg, (days −9 to −6), as well as Cytoxan, 50mg/kg, days −5 to −2, Cotrimoxazol, 10 mg/kg, days −10 to −2,Allopurinol, 300 mg/kg, days −10 to −1 and Ara-C, 25 mg intrathecally.ASCT consisted of non-purged bone marrow cells, and 0.79×10⁸ nucleatedcells/kg were infused IV on day 0. On day +1, the patient received PBLfrom an HLA-matched donor at an equivalent dose of 10⁷ T cells/kg. Onday +1, she also received 6×10⁶ IU of rhIL-2/m² by subcutaneousinjection. No GVHD was observed.

[0111] Patient No. 4.

[0112] This male patient was diagnosed with AML, FAB M2, and was infirst complete remission at the time of ASCT. The patient was 23 yearsold at the time of ASCT. Prior to ASCT, he received a conditioningregimen of Busulfan 4 mg/kg, days −9 to −6, Cytoxan 60 mg/kg, days −3 to−2, Thiotepa 5 mg/kg/day, days −5 to −4, Cotrimoxazol, 10 mg/kg, days−10 to −2, Allopurinol, 300 mg/kg, days −10 to −1 and Ara-C, 25 mgintrathecally. ASCT consisted of non-purged 1.37×10⁸ nucleated bonemarrow cells/kg, infused IV on day 0. On day +1, the patient receivedPBL from an HLA-matched donor at an equivalent dose of 10⁷ T cells/kg.On day +1, he also received 6×10⁶ IU of rhIL-2/m² by subcutaneousinjection. GVHD was suspected (Grade I) in the skin.

[0113] Patient No. 5.

[0114] This male patient was diagnosed with chronic myelogenous leukemia(CML) in chronic phase. The original CML karyotype was positive for thePhiladelphia chromosome (Ph+). The patient was in chronic phase (CP) andwas Ph− at the time of ASCT. The patient was 51 years old at the time ofASCT. Prior to ASCT, he received a conditioning regimen of total bodyirradiation (TBI) 200 cGY/day, days −5 to −3, and Cytoxan 60mg/kg, days−2 to −1. ASCT consisted of non-purged 0.5×10⁸ nucleated bone marrowcells/kg, infused IV on day 0.

[0115] On day +71, as soon as blood counts had stabilized (WBC:4,200/mm³; Hb: 11.5 g %; platelets: 133,000/mm³), the patient received3×10⁷ T cells/kg of PBL from an HLA-matched brother. No GVHD wasobserved; hence, the allo-CMI regimen was escalated. On day +10⁷, thepatient received 4.6×10⁷ T cells/kg of PBL from the same donor. Startingon day +10⁷, he also received 6×10⁶ IU of rhIL-2/m²/day for 3 days, bysubcutaneous injection. No GVHD was observed. On day +240, after the Ph+karyotype had reappeared, the patient received 4.95×10⁷ cells/kg ofcytokine-activated lymphocytes (“CAL”) from the same donor. Starting onday +240, he also received 6×10⁶ IU of rhIL-2/m²/day for 3 days, bysubcutaneous injection. The CAL were produced by exposing the donor'sPBL to 6,000 IU/ml rhIL-2 for four days in culture.

[0116] Patient No. 6.

[0117] This female patient was diagnosed with AML, FAB M2, and was infirst complete remission at the time of ASCT. The patient was 50 yearsold at the time of ASCT. Prior to ASCT, she received a conditioningregimen of Busulfan 4 mg/kg, days −9 to −6, Cytoxan 60 mg/kg, days −3 to−2, Thiotepa 5 mg/kg/day, days −5 to −4, Cotrimoxazol, 10 mg/kg, days−10 to −2, Allopurinol, 300 mg/kg, days −10 to −1 and Ara-C, 25 mgintrathecally. ASCT consisted of non-purged 0.64×10⁸ nucleated bonemarrow cells/kg infused IV on day 0. On day +58, as soon as blood countshad stabilized (WBC: 4,400/mm³; Hb: 9.5 g %; platelets: 66,000/mm³), thepatient received 5×10⁷ T cells/kg of PBL from an HLA-matched sister. Onday +86 the patient received a second dose of 6.1×10⁷ T cells/kg of PBLfrom the HLA-matched sister. Starting on day +86, she also received6×10⁶ IU of rhIL-2/m²/day for 3 days, by subcutaneous injection. No GVHDwas observed.

[0118] Patient No. 7.

[0119] This male patient was diagnosed with CML. The original CMLkaryotype was Ph+. The patient was in chronic phase (CP) and 50% of hismarrow cells were Ph+ (i.e., the patient was Ph+) at the time of ASCT.The patient was 47 years old at the time of ASCT. Prior to ASCT, hereceived a conditioning regimen of TBI 200 cGY/day, days −5 to −3, andCytoxan 60mg/kg, days −2 to −1. ASCT consisted of non-purged 0.98×10⁸nucleated bone marrow cells/kg, infused IV on day 0. On day +55, as soonas blood counts had stabilized (WBC: 6,900/mm³; Hb: 12.0 g %; platelets:248,000/mm³), the patient received 4×10⁷ T cells/kg of PBL from anHLA-matched sister. No GVHD developed. On day +77, the patient received2.8×10⁷ cells/kg of PBL from the HLA-matched sister. Starting on day+77, he also received 6×10⁶ IU of rhIL-2/m²/day for 3 days, bysubcutaneous injection. No GVHD was observed.

[0120] Patient No. 8.

[0121] This male patient was diagnosed with non-Hodgkin's lymphoma(NHL), Burkitt-like, and was in a second partial remission at the timeof ASCT. As used herein, the term “partial remission” indicates at leasta 50% response (i.e., at least a 50% reduction of lymphoma cell mass)but with continued evidence of disease. The patient was 36 years old atthe time of ASCT. Prior to ASCT, he received a conditioning regimen ofetoposide, 200 mg/M²/day, days −6 to −3, thiotepa, 40 mg/m²/day, days −5to −2, Ara-C, 200 mg/m²/day, days −4 to −1, Cytoxan, 60 mg/kg/day, day−3, and melphalan, 60 mg/m²/day, days −2 to −1.

[0122] ASCT consisted of 0.74×10⁸/kg viable bone marrow nucleated cellsplus 2.36×10⁸/kg viable peripheral blood stem cells. SubcutaneousGM-CSF, 5 ug/kg/day, was administered from day +1 to day +18. Prior toASCT, the autologous cells were purged with Dynal magnetic beads coatedwith anti-CD19 for elimination of residual lymphoma cells.

[0123] On day +90, the patient received 5×10⁷ cells/kg of PBL from anHLA-matched brother. The patient showed no signs of GVHD following thisfirst cell infusion. Polymerase chain reaction (PCR) analysis using twoVNTR loci (VNTR Variable Number of Tandem Repeats) revealed no evidenceof circulating donor-specific cells. On day +124, the patient received5×10⁷ cells/kg of PBL from the same donor. This was followed by threedays outpatient treatment with 6×10⁶ IU of rhIL-2/m²/day, bysubcutaneous injection, beginning on day +124.

[0124] Fifty days later (day +174) the patient developed pancytopenia,and bone marrow biopsy revealed severe hypocellular marrow withincreased numbers of large granular lymphocytes and plasma cells.Lymphocytes with a similar morphology were found in the blood. RepeatedPCR using two different VNTR loci showed partial engraftment of donorcells on day +191 and 100% engraftment of donor cells on day +211. Anallogeneic BMT was then performed by infusing the patient with3.4×10⁸/kg of the donor's marrow cells; no post-transplant anti-GVHDprophylaxis was administered. The patient had rapid three-lineageengraftment with normal platelet counts after 14 days and normalhemoglobin after 26 days. WBC normalized after 10 days with 70%neutrophils, 5% monocytes and 25% lymphocytes. Large granularlymphocytes disappeared from the blood. On day 14 following allogeneicBMT, the patient showed minimal signs of acute GVHD with involvement ofskin and oral cavity. There was no intestinal or liver involvement.Since then the patient has continued to experience grade I/IImucocutaneous GVHD, partially controlled with steroids and cyclosporinA.

[0125] Patient No. 9.

[0126] This male patient was diagnosed with NHL, follicular mixed IV A,and was in a second partial remission at the time of ASCT. The patientwas 39 years old at the time of ASCT. Prior to ASCT, he received aconditioning regimen of etoposide, 200 mg/M²/day, days −6 to −3;thiotepa, 40 mg/m²/day, days −5 to −2; Ara-C, 200 mg/m²/day, days −4 to−1; Cytoxan, 60 mg/kg/day, day −3; and melphalan, 60 mg/m²/day, days −2to −1.

[0127] ASCT consisted of 0.5×10⁸ nucleated bone marrow cells/kg, infusedIV on day 0. Prior to ASCT, the autologous cells were purged with Dynalmagnetic beads coated with anti-CD19 for elimination of contaminatingtumor cells.

[0128] At week 8 post-ASCT, the patient received 5×10⁷ T cells/kg of PBLfrom an HLA-matched sister. At week 12 post-ASCT, the patient received5×10⁷ T cells/kg of PBL from the same donor. Starting at week 12, on thesame day as the administration of the second Allo-CMI treatment, thepatient also received 6×10⁶ IU of rhIL-2/m²/day for 3 days, bysubcutaneous injection. No GVHD was observed. At week 16 post-ASCT, thepatient received 0.5×10⁷ CAL/kg from the HLA-matched sister. The CALwere produced by exposing the donor's PBL to 6,000 IU/ml rhIL-2 for fourdays in culture. No GVHD was observed.

[0129] Patient No. 10.

[0130] This female patient was diagnosed with NHL, mixed cellularity IIA, and was in a second complete remission at the time of ASCT. Thepatient was 36 years old at the time of ASCT. Prior to ASCT, shereceived a conditioning regimen of etoposide, 200 mg/m²/day, days −6 to−3, thiotepa, 40 mg/m²/day, days −5 to −2, Ara-C, 200 mg/m²/day, days −4to −1, Cytoxan, 60 mg/kg/day, day −3, and melphalan, 60 mg/m²/day, days−2 to −1.

[0131] ASCT consisted of 0.94×10⁸ non-purged nucleated bone marrowcells/kg plus 3.9×10⁷ peripheral blood stem cells mobilized by G-CSFprior to collection with the CS 3000® Plus blood cell separator (BaxterHealthcare Corporation, Fenwal System Catalogue No. 4R4538). Cells wereinfused IV on day 0.

[0132] At week 10 post-ASCT, as soon as blood counts had stabilized(WBC: 4,300/mm³; Hb: 11.2 g %; platelets: 116,000/mm³), the patientreceived 3×10⁷ T cells/kg of PBL from an HLA-matched brother. At week 15post-ASCT, the patient received 4.1×10⁷ T cells/kg of PBL from the samedonor. Starting at week 15, on the same day as the administration of thesecond Allo-CMI treatment, the patient also received 6×10⁶ IU ofrhIL-2/m²/day for 3 days, by subcutaneous injection. At week 23post-ASCT, the patient received 5×10⁷ CAL/kg from the HLA-matchedbrother. The CAL cells were produced by exposing the donor's PBL to6,000 IU/ml rhIL-2 for four days in culture. Starting at week 23, on thesame day as the administration of the Allo-CCI treatment, the patientalso received 6×10⁶ IU of rhIL-2/m²/day for 3 days, by subcutaneousinjection. No GVHD was observed.

[0133] Patient No. 11.

[0134] This female patient was diagnosed with NHL, immunoblastic, andwas in a second complete remission at the time of ASCT. The patient was21 years old at the time of ASCT. Prior to ASCT, she received aconditioning regimen of etoposide, 200 mg/m²/day, days −6 to −3;thiotepa, 40 mg/m²/day, days −5 to −2; Ara-C, 200 Mg/M2/day, days −4 to−1; Cytoxan, 60 mg/kg/day, day −3; and melphalan, 60 mg/M²/day, days −2to −1.

[0135] ASCT consisted of 3.82×10⁸ non-purged bone marrow cells/kg plus1.29×10⁸ peripheral blood stem cells mobilized with G-CSF prior tocollection with the CS 3000® Plus blood cell separator. Cells wereinfused IV on day 0.

[0136] At week 10 post-ASCT, the patient received 5×10⁷ T cells/kg ofPBL from an HLA-matched sister. At week 15 post-ASCT, the patientreceived a second infusion of 5×10⁷ T cells/kg of PBL from theHLA-matched sister. At week 19 post-ASCT, the patient received a thirdinfusion of 5×10⁷ T cells/kg of PBL from the same donor. No GVHD wasobserved, but the patient did not accept the suggestion that she receivein vivo rhIL2. At week 30 post-ASCT, the patient received a fourthinfusion of 5×10⁷ T cells/kg of PBL from the HLA-matched sister.Starting at week 23, on the same day as the administration of the fourthAllo-CMI treatment, the patient also received 6×10⁶ IU of rhIL-2/m²/dayfor 3 days, by subcutaneous injection. No GVHD has been observed todate.

[0137] Patient No. 12.

[0138] This female patient was diagnosed with NHL, diffuse large cell,and was in a condition of relapse at the time of ASCT. The patient was21 years old at the time of ASCT. Prior to ASCT, she received aconditioning regimen of etoposide, 200 mg/m²/day, days −6 to −3;thiotepa, 40 mg/m²/day, days −5 to −2; Ara-C, 200 mg/m²/day, days −4 to−1; Cytoxan, 60 mg/kg/day, day −3; and melphalan, 60 mg/m²/day, days −2to −1.

[0139] ASCT consisted of 1.8×10⁸ non-purged mononuclear bone marrowcells/kg, infused IV on day 0.

[0140] At week 6 post-ASCT, as soon as blood counts had stabilized (WBC:4,400/mm³; Hb: 11.7 g %; platelets: 150,000/mm³), the patient received5×10⁷ T cells/kg of PBL from a single locus-mismatched sister. At week10 post-ASCT, the patient received a second infusion of 5×10⁷ T cells/kgof PBL from the same donor. Starting at week 10, on the same day as theadministration of the second Allo-CMI treatment, the patient alsoreceived 6×10⁶ IU of rhIL-2/m²/day for 3 days, by subcutaneousinjection. No GVHD was observed.

[0141] Patient No. 13.

[0142] This male patient was diagnosed with AML, FAB M5, and was infirst complete remission at the time of ASCT. The patient was 46 yearsold at the time of ASCT. Prior to ASCT, he received a conditioningregimen of Busulfan 4 mg/kg, days −9 to −6; Cytoxan 60 mg/kg, days −3 to−2; Thiotepa 5 mg/kg/day, days −5 to −4; Cotrimoxazol, 10 mg/kg, days−10 to −2, Allopurinol, 300 mg/kg, days −10 to −1 and Ara-C, 25 mgintrathecally. ASCT consisted of 1.39×10⁸ non-purged bone marrow cellsinfused IV on day 0. At week 11, the patient received 3.86×10⁷ Tcells/kg of PBL from an HLA-matched brother. No GVHD developed.

[0143] Patient No. 14.

[0144] This female patient was diagnosed with AML, FAB M3, and was insecond partial remission at the time of ASCT. The patient was 12 yearsold at the time of ASCT. Prior to ASCT, she received a conditioningregimen of Busulfan 4 mg/kg, days −9 to −6; Cytoxan 60 mg/kg, days −3 to−2; Mitoxantrone 6 mg/m²/day, days −5 to −4; Cotrimoxazol, 10 mg/kg,days −10 to −2, Allopurinol, 300 mg/kg, days −10 to −1 and Ara-C, 25 mgintrathecally. ASCT consisted of 1.14×10⁸ non-purged marrow cellsinfused IV on day 0. At week 16, the patient received 2×10⁷ T cells/kgof single locus-mismatched PBL from her mother. At week 20, the patientreceived 1000 mg/m² Cytoxan in order to improve the efficacy andtemporal engraftment of a second infusion of the donor's PBL. Twentyfour hours later, the patient received 5×10⁷ T cells/kg from the samedonor. Starting on the same day as the administration of the secondAllo-CMI treatment, the patient also received 6×10⁶ IU of rhIL-2/m²/dayfor 3 days, by subcutaneous injection. No GVHD has been noted thus far.

[0145] Patient No. 15.

[0146] This male patient was diagnosed with AML, FAB MS, and was infirst complete remission at the time of ASCT. The patient was 6½ yearsold at the time of ASCT. Prior to ASCT, he received a conditioningregimen of Busulfan 4 mg/kg, days −9 to −6; Cytoxan 60 mg/kg, days −3 to−2; Thiotepa 5 mg/kg/day, days −5 to −4; Cotrimoxazol, 10 mg/kg, days−10 to −2, Allopurinol, 300 mg/kg, days −10 to −1 and Ara-C, 25 mgintrathecally. ASCT consisted of 2.7×10⁸ non-purged marrow cells infusedIV on day 0. At week 14, the patient received 5×10⁷ T cells/kg of singlelocus-mismatched PBL from his father. No GVHD has developed, and thepatient is scheduled to receive a second dose of allo-CMI plus in vivorhIL2.

[0147] Patient No.16.

[0148] This male patient was diagnosed with NHL, mixed large and smallcell, in the lymph nodes, in February of 1993. There was also heavy bonemarrow involvement. He received chemotherapy (12 courses of MACOP-B),but relapsed in the lymph nodes and bone marrow in October of 1993. Heunderwent additional chemotherapy and was in a second partial remissionat the time of ASCT, with recurrence in the marrow. The patient was 45years old at the time of ASCT. Prior to ASCT, he received a conditioningregimen consisting of etoposide, 200 mg/m²/day, days −6 to −3; thiotepa,40 mg/m²/day, days −5 to −2; Ara-C, 200 mg/m²/day, days −4 to −1;Cytoxan, 60 mg/kg/day, day −3; and melphalan, 60 mg/m²/day, days −2 to−1.

[0149] ASCT consisted of 2.15×10⁸ non-purged G-CSF-mobilized peripheralblood stem cells, infused IV on day 0.

[0150] At week 7 post-ASCT, the patient received 3×10⁷ T cells/kg of PBLfrom an HLA-matched sister. At week 11 post-ASCT, the patient received asecond infusion of 3×10⁷ T cells/kg of PBL from the same donor. Startingat week 11, on the same day as the administration of the second Allo-CMItreatment, the patient also received 6×10⁶ IU of rhIL-2/m2/day for 3days, by subcutaneous injection. No GVHD has been observed to date.

[0151] Patient No. 17.

[0152] This female patient was diagnosed with metastatic breast cancer,with metastases to the liver and spine. She underwent a right partialmastectomy with axillary lymph node dissection showing involvement of18/35 lymph nodes. The patient was 43 years old at the time of ASCT.Prior to ASCT, she received a conditioning regimen of carboplatin, 200mg/m²/day, days −7. to −4; thiotepa, 60 mg/m²/day, days −6 to −4;etoposide, 200 Mg/m²/day, days −5 to −3; and melphalan, 60 mg/m²/day,days −4 to −3.

[0153] ASCT consisted of 3.7×10⁸ peripheral blood stem cells cells/kg,infused IV on day 0. The stem cells were collected after mobilizationwith G-CSF (7.5 mg/kg/day for 5 days) using three collections with theCS 3000® Plus blood cell separator. Following ASCT, the patientrecovered with no complications and was discharged on day 20 post-ASCT.

[0154] A fully HLA-matched (A, B, DR and mixed lymphocyte reaction(MLR)-negative) sibling was chosen as donor of PBL for Allo-CMI. At week8 post-ASCT, as soon as the patient achieved a stable clinical condition(WBC: 2,700/mm³; Hb: 9.1 g %; platelets: 56,000/mm³), the patientreceived 5×10⁷ T cells/kg of PBL from the HLA-matched sibling. Startingat week 8, on the same day as the administration of the first Allo-CMItreatment, the patient also received 6×10⁶ IU of rhIL-2/m2/day for 3days, by subcutaneous injection. No GVHD has been observed to date.

[0155] Patient No. 18.

[0156] This male patient was diagnosed with metastatic breast cancer,with metastases to the bone marrow, sternum and vertebrae T₄ to T₇. Heunderwent lumpectomy with axillary lymph node dissection showinginvolvement of 16/18 lymph nodes. The patient was 36 years old at thetime of ASCT. Prior to ASCT, he received a conditioning regimen ofcarboplatin, 200 mg/m/dlay, days −7 to −4; thiotepa, 60 mg/m²/day, days−6 to −4; etoposide, 200 mg/m²/day, days −5 to −3; and melphalan, 60mg/M²/day, days −4 to −3.

[0157] ASCT consisted of 1.81×10⁸ peripheral blood stem cells cells/kg,infused IV on day 0. The stem cells were collected after mobilizationwith G-CSF (7.5 mg/kg/day for 5 days) using three collections with theCS 3000® Plus blood cell separator. Following ASCT, the patientrecovered with no complications and was discharged.

[0158] A fully HLA-matched (A, B, DR and mixed lymphocyte reaction(MLR)-negative) brother was chosen as donor of PBL for Allo-CMI. At week5 post-ASCT, as soon as the patient achieved a stable clinical condition(WBC: 11,000/mm³; Hb: 11.5 g %; platelets: 201,000/mm³), the patientreceived 2.3×10⁷ T cells/kg of PBL from the HLA-matched brother.Starting at week 5, on the same day as the administration of the firstAllo-CMI treatment, the patient aIlso received 6×10⁶ IU of rhIL-2/m²/dayfor 3 days, by subcutaneous injection. No GVHD has been observed todate.

[0159] Portions of the above-described patient treatment protocols aresummarized in Table 1, below. Patients are grouped according to disease(AML, CML, NHL and breast cancer). TABLE 1^(a) Patient Status Allo CellsNo. Pre-ASCT Cond. Regimen Type ASCT (T Cells/Kg) AML:  1 1CR BU/CYABMT: Purged PBL 10⁴ − 10⁷ (day +1 to +47)  2 1CR 13U/CY ABMT: PurgedPBL 10⁴ − 10⁷ (day +1 to +80)  3 1CR BU/CY ABMT: Non-Purged PBL 10⁷ +IL2 (day +1)  4 1CR BU/CY + TT ABMT: Non-Purged PBL 10⁷ + IL2 (day +1) 6 1CR BU/CY + TT ABMT: Non-Purged 1. PBL 2. PBL + IL2 13 1CR BU/CY + TTABMT: Non-Purged 1. PBL 14 2PR BU/CY + MX ABMT: Non-Purged 1. PBL 2.Cytoxan/PBL + IL2 15 1CR BU/CY + TT ABMT: Non-Purged 1. PBL CML:  5CP(Ph⁻) CY/TBI ABMT: Non-Purged 1. PBL 2. PBL + IL2 3. CAL + IL2  7CP(Ph⁺) CY/TBI ABMT: Non-Purged 1. PBL 2. PBL + IL2 NHL:  8 2PR ETACMABMT + PBSC: Purged 1. PBL 2. PBL + IL2 3. Allogeneic BMT (due to marrowaplasia)  9 2PR ETACM ABMT: Purged 1. PBL 2. PBL + IL2 3. CAL + IL2 102CR ETACM ABMT + PBSC: Non-Purged 1. PBL. 2. PBL + IL2 3. CAL + IL2 112CR ETACM ABMT + PBSC: Non-Purged 1. PBL 2. PBL 3. PBL 4. PBL + IL2 12Relapse ETACM ABMT: Non-Purged 1. PBL 2. PBL + IL2 16 2PR ETACM ABMT +PBSC: Non-Purged 1. PBL BREAST CANCER: 17 Metastatic CTEM PBSC:Non-Purged 1. PBL + IL2 18 Metastatic CTEM PBSC: Non-Purged 1. PBL + IL2

[0160] II. Results

[0161] The above-described patients have been followed for variouslengths of time. The disease status of the patients is summarized below:

[0162] Patient No. 1 relapsed after 6 months and died at 9 months afterASCT.

[0163] Patient No. 2 relapsed after 12 months and died at 19 monthsafter ASCT.

[0164] Patient No. 3 is alive and well over 34 months after ASCT.

[0165] Patient No. 4 is alive and well over 24 months after ASCT.

[0166] Patient No. 5 is alive and well over 23 months after ASCT,showing no signs of the Philadelphia chromosome (Ph-) by cytogeneticanalysis and being negative by PCR for the bcr/abl translocationcharacteristic of the Philadelphia chromosome. The patient is receiving9×10⁶ units of IFNα (Roferon A, S.C.) every day.

[0167] Patient No. 6 is alive and well over 23 months after ASCT with noevidence of disease.

[0168] Patient No. 7 is alive and well over 21 months post-ASCT.Currently, the patient is on IFINα treatment, and is hematologicallynormal with no clinical sign of disease.

[0169] Patient No. 8, currently at nearly 2 years after initial ASCT andover one year following allogeneic BMT, is alive and well with noevidence of lymphoma. The patient has mild cutaneous chronic GVHD withKarnofsky score of 100%

[0170] Patient No.9 was alive and well until 18 months after ASCT. At 19months he has lymphadenopathy and is being evaluated for relapse.

[0171] Patient No. 10 is alive and well over 15 months after ASCT withno evidence of disease.

[0172] Patient No. 11 is alive and well over 13 months after ASCT withno evidence of disease.

[0173] Patient No. 12 underwent relapse at 3 months post-ASCT and diedat 5 months post-ASCT.

[0174] Patient No. 13 underwent relapse at 4 months post-ASCT, beforereceiving allo-CMI with rhIL2. He is alive and further immunotherapy isplanned.

[0175] Patient No. 14 is alive and well with no evidence of disease over5 months post-ASCT.

[0176] Patient No. 15 is alive and well with no evidence of disease over4 months post-ASCT.

[0177] Patient No. 16 is alive and well with no evidence of disease 3months post-ASCT.

[0178] Patient No. 17 is alive and well with no evidence of disease 2months post-ASCT.

[0179] Patient No. 18 is alive and well with no evidence of disease 1.5months post-ASCT.

[0180] The results reported above indicate that Allo-CMI and Allo-CCImay be the most rational and practical approaches for eradication ofresidual malignant cells. GVT effects induced by administration ofallogeneic lymphocytes may be further enhanced by administration in vivoof rhIL2.

EXAMPLE 3 Utility of Cytokines Other than IL2

[0181] Cytokines used in the following experiments were obtained asfollows: (1) rhIL2 was provided by Dr. C. R. Franks (EuroCetus BY,Amsterdam, The Netherlands) as a lyophilized powder in 1 mg vialscontianing 18×10⁶ international units (IU). (2) Recombinantinterferon-gamma (rIFNγ) was provided by Roussel Uclaf (Romainville,France) as a lyophilized powder containing 2×10⁷ U/mg. (3) Recombinanthuman IL-6 (rIL-6) was kindly provided by Dr. M. Revel and Dr. O. Laub(InterPharm Laboratories, Rehovot, Israel) in a concentration of 1.13mg/ml protein containing 43×10⁶ IU/ml. (4) Recombinant human IL-7(rhIL-7) was provided by Pepro Tech (New Jersey, USA) as lyophilizedpowder and was reconstituted to 100 mg/ml.

[0182] Lymphocytes were preactivated in vitro with rhIL2 or withcombinations of rhIL2 and other cytokines for four days at 37° C.Concentrations of cytokines used for in vitro incubation of lymphocyteswere as follows: (a) rhIL2: 6000 IU/ml; (b) rIL6: 100-1000 U/ml; (c)rIL7: long/ml; and (d) IFNy: 1000 U/ml. Anti-tumor effects of CALcollected post-culturing were assayed against natural killer(NK)-sensitive K562 tumor cells and NK-resistant Daudi tumor cells. Thein vitro toxicity was evaluated by specific chromium release followingincubation of effector cells with chromium-labeled tumor cells. Resultsare presented below as lytic units per 10⁶ effector cells, determinedfor 30% lysis of 5×10³ target cells: Cytotoxicity (Lytic Units/10⁶Cells) Anti-Daudi Anti-K562 IL2 alone 48 53 IL2 + IL6 + IL7 + IFNγ 210129

What is claimed is:
 1. A method of treating a human cancer patient, saidpatient having undergone a malignant cell debulking procedure and beingat risk for disease relapse due to a population of residual malignantcells that may remain viable in said patient following said debulkingprocedure, comprising: a) providing a sample of stem cells from saidpatient, said sample being suitable for autologous transplantation intosaid patient; b) performing an autologous transplant of said patientwith said sample; c) monitoring said patient until said patient ispartially hematopoiesis recovered but is not fully immune-reconstituted;d) administering to said patient an HLA-compatible, allogeneicperipheral blood leukocyte preparation having lymphocytes, in a regimenthat causes a clinically significant graft-versus-malignant cellresponse; and e) monitoring said patient for levels of malignant cellsderiving from said population.
 2. The method of claim 1, wherein saidregimen comprises the following steps in sequence: a) treating saidpatient by administration of about 10⁷ cells/kg to about 10⁹ cells/kgofHLA-compatible, allogeneic peripheral blood lymphocytes; b) monitoringsaid patient for indications of a graft-versus-malignant cell response;and c) if no or insufficient graft-versus-malignant cell responsedevelops in said patient, escalating said treatment by performing atleast one procedure selected from the group consisting of (1)administration of a number of HLA-compatible, allogeneic peripheralblood lymphocytes greater than the number of lymphocytes administered instep (a); (2) administration of a number of HLA-compatible, allogeneicperipheral blood lymphocytes at least as great as the number oflymphocytes administered in step (a), accompanied by administration ofat least one T-cell-activating cytokine to said patient; (3)administration of HLA-compatible, allogeneic CAL's to said patient; and(4) administration of HLA-compatible, allogeneic CAL's, accompanied byadministration in vivo of at least one T-cell-activating cytokine tosaid patient; wherein more than one of said procedures is performed ifno or insufficient graft-versus-malignant cell response develops in saidpatient following said first or subsequent procedure.
 3. The method ofclaim 2, wherein step (a) further comprises administration in vivo of atleast one T-cell-activating cytokine to said patient.
 4. A method oftreating a human cancer patient, said patient having undergone amalignant cell debulking procedure and being at risk for disease relapsedue to a population of residual malignant cells that may remain viablein said patient following said debulking procedure, comprising: a)providing a sample of stem cells from said patient, said sample beingsuitable for autologous transplantation into said patient; b) performingan autologous transplant of said patient with said sample; c) monitoringsaid patient until said patient is partially hematopoiesis recovered butis not fully immune-reconstituted; d) administering to said patient anHLA-compatible, allogeneic peripheral blood leukocyte preparation havinglymphocytes, in a regimen that causes a mild graft-versus-host response;and e) monitoring said patient for levels of malignant cells derivingfrom said population.
 5. The method of claim 4, wherein said regimencomprises the following steps in sequence: a) treating said patient byadministration of about 10⁷ cells/kg to about 10⁹ cells/kg ofHLA-compatible, allogeneic peripheral blood lymphocytes; b) monitoringsaid patient for indications of a mild graft-versus-host response; andc) if no or insufficient graft-versus-host response develops in saidpatient, escalating said treatment by performing at least one procedureselected from the group consisting of (1) administration of a number ofHLA-compatible, allogeneic peripheral blood lymphocytes greater than thenumber of lymphocytes administered in step (a); (2) administration of anumber of HLA-compatible, allogeneic peripheral blood lymphocytes atleast as great as the number of lymphocytes administered in step (a),accompanied by administration of at least one T-cell-activating cytokineto said patient; (3) administration of HLA-compatible, allogeneic CAL'sto said patient; and (4) administration of HLA-compatible, allogeneicCAL's, accompanied by administration of at least one T-cell-activatingcytokine to said patient; wherein more than one of said procedures isperformed if no or insufficient graft-versus-host response develops insaid patient following said first or subsequent procedure.
 6. The methodof claim 5, wherein step (a) further comprises administration in vivo ofat least one T-cell-activating cytokine to said patient.
 7. The methodof claim 4, wherein said regimen comprises the following steps insequence: a) administering to said patient about 10⁷ cells/kg to about10⁹ cells/kg of HLA-compatible, allogeneic peripheral blood lymphocytesand at least one T-cell-activating cytokine to said patient; b)monitoring said patient for signs of a mild graft-versus-host response;c) if no or insufficient graft-versus-host response develops in saidpatient, administering about 10⁷ cells/kg to about 10⁹ cells/kg ofHLA-compatible, allogeneic CAL and at least one T-cell-activatingcytokine to said patient; and d) monitoring said patient for signs of amild graft-versus-host response.
 8. The method of claim 4, wherein saidregimen comprises the following steps in sequence: a) administering tosaid patient about 10⁵ cells/kg to about 10⁹ cells/kg of HLA-compatible,allogeneic peripheral blood lymphocytes, said HLA-compatible, allogeneicperipheral blood lymphocytes comprising CAL, and at least oneT-cell-activating cytokine to said patient; b) monitoring said patientfor signs of a mild graft-versus-host response; c) if no or insufficientgraft-versus-host response develops in said patient, administering about10⁵ cells/kg to about 10⁹ cells/kg of HLA-compatible, allogeneic CAL andat least one T-cell-activating cytokine to said patient; and d)monitoring said patient for signs of a mild graft-versus-host response.9. The method of claim 2, 3, 5, 6, 7 or 8 wherein said cytokine isselected from the group consisting of IL2, IL4, IL5, IL6, IL7, IFNα,IFNγ and TNFα.
 10. The method of claim 4, wherein said stem cells areobtained from bone marrow.
 11. The method of claim 4, wherein said stemcells are obtained from the peripheral circulation.
 12. The method ofclaim 4, wherein said stem cells are obtained from fetal sourcesselected from the group consisting of fetal tissue, fetal circulationand umbilical cord blood.
 13. The method of claim 4, wherein saidmalignant cells are leukemia cells.
 14. The method of claim 4, whereinsaid malignant cells are lymphoma cells.
 15. The method of claim 4,wherein said malignant cells are breast cancer cells.
 16. The method ofclaim 1 or 4, wherein said HLA-compatible cells are fully HLA-matchedwith said patient.
 17. The method of claim 1 or 4, wherein saidHLA-compatible cells are at least haploidentical with said patient. 18.The method of claim 1 or 4, wherein said HLA-compatible cells are singleHLA locus-mismatched cells from a sibling of said patient.
 19. Anarticle of manufacture comprising packaging material and a biologicalcell container within said packaging material, wherein said packagingmaterial contains a label or package insert indicating that saidbiological cell container and any contents therein are to be used in themethod of claim 1 or 4.